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23  WEST  MAIN  STREET 

WEBSTER,  N.Y.  14SM 

(716)E72.4S03 


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CIHM/ICMH 

Microfiche 

Series. 


CIHM/ICMH 
Collection  de 
microfiches. 


Canadian  Institute  for  Hictorical  Microreproductions  /  institut  Canadian  de  microreproductions  hisioriques 


Technical  and  Bibliographic  Notea/Notas  tachniquas  at  bibliographiquas 


The  Institute  has  attomptad  to  obtain  the  best 
original  copy  available  for  fil.-ning.  Features  of  this 
copy  which  may  be  bibliographically  unique, 
which  may  alter  any  of  ^he  images  in  the 
reproduction,  or  which  may  significantly  change 
the  usua!  method  of  filming,  are  checked  below. 


□    Coloured  covers/ 
Couverture  de  couleur 

□    Covers  damaged/ 
Couverture  endommagtfte 

□    Covers  restored  and/or  laminated/ 
Couverture  restaur^e  et/ou  pellicul6e 


D 


D 
D 


n 


Cover  title  missing/ 

Le  titre  de  couverture  manque 


I      I    Coloured  maps/ 


Cartes  gdographiques  en  couleur 


□    Coloured  ink  (i.e.  other  than  blue  or  black)/ 
Encre  de  couleur  (i.e.  autre  que  bleue  ou  noire) 

□    Coloured  plates  and/or  illustrations/ 
Planches  et/ou  illustrations  en  couleur 


D 


Bound  with  other  material/ 
Relid  avec  d'autres  documents 

Ti'^ht  binding  may  causa  shadows  or  distortion 
along  interior  margin/ 

La  reliure  serrie  paut  causer  de  I'ombre  ou  de  la 
distortion  le  long  de  la  marge  int6riaure 

Blank  leaves  added  during  restoration  may 
appear  within  the  text.  Whenever  possible,  these 
have  been  omitted  from  filming/ 
II  se  peut  que  certaines  pages  blanches  ajout^as 
lors  d'une  restauration  apparaissant  dans  le  taxte, 
mais,  lorsqua  cela  itait  possible,  ces  pages  n'ont 
pas  6t6  film6es. 

Additional  comments:/ 
Commentaires  suppl6mentaires; 


L'Institut  a  microfilm^  le  meilleur  exemplaire 
qu'il  lui  a  6t6  possible  de  se  procurer.  Les  details 
da  cat  exemplaire  qui  sont  peut-dtre  uniques  du 
point  de  vue  bibliographique,  qui  peuvent  modifier 
une  image  reproduite,  ou  qui  peuvent  exiger  une 
modification  dans  la  m6thode  normale  de  filmage 
sont  indiqu6s  ci-dessous. 


I     1    Coloured  pages/ 


D 


Pages  de  couleur 

Pages  damaged/ 
Pages  endommagdes 


□    Pages  restored  and/or  laminated/ 
Pages  restaur6es  et/ou  pelliculdes 

I      I    Pages  discoloured,  stained  or  foxed/ 


Pages  d6color6es,  tachet6es  ou  piqu^es 

Pages  detached/ 
Pages  ddtach^es 

Showthrough/ 
Transparence 

Quality  of  prir 

Qualit^  in^gale  de  I'impression 

Includes  supplementary  materi{ 
Comprend  du  materiel  supplementaire 

Only  edition  available/ 
Soule  Edition  disponible 


I      I  Pages  detached/ 

Fyj  Showthrough/ 

I      I  Quality  of  print  varies/ 

I      I  Includes  supplementary  material/ 

I      I  Only  edition  available/ 


Pages  wholly  or  partially  obscured  by  errata 
slips,  tissut^s,  etc.,  have  been  refilmed  to 
ensure  the  best  possible  image/ 
Les  pages  totalement  ou  partiellement 
obscurcies  par  un  feuillet  d'errata,  une  pelure, 
etc.,  ont  6x6  film^es  6  nouveau  de  fa^on  6 
obtenir  la  meilleure  image  possible. 


This  item  is  filmed  at  the  reduction  ratio  checked  below/ 

Ce  document  est  film6  au  taux  de  rMuction  indiquA  ci-dessous. 


10X 

14X 

18X 

22X 

28X 

30X 

V 

1 

1 

1 

12X 

16X 

20X 

24X 

28X 

32X 

The  copy  filmed  here  has  been  reproduced  thanks 
to  the  generosity  of: 

Thomai  Fisher  Rare  Booic  Library, 
University  of  Toronto  Library 

The  images  appearing  here  are  the  best  quality 
possible  considering  the  condition  and  legibility 
of  the  original  copy  and  in  keeping  with  the 
filming  contract  specifications. 


L'exemplaire  filmi  fut  reproduit  grftce  A  la 
g^nArositA  de: 

Thomas  Fisher  Rare  Book  Library, 
University  of  Toronto  Library 

Les  images  suivantes  ont  6t6  reproduites  avec  le 
plus  grand  soin,  compte  tenu  de  la  condition  et 
de  la  nettet6  de  l'exemplaire  film6.  et  en 
conformity  avec  les  conditions  du  contrat  de 
filmage. 


Original  copies  in  printed  paper  covers  are  filmed 
beginning  with  the  front  cover  and  ending  on 
the  last  page  with  a  printed  or  illustrated  impres- 
sion, or  the  back  cover  when  appropriate.  All 
other  original  copies  are  filmed  beginning  on  the 
first  page  with  a  printed  or  illustrated  impres- 
sion, and  ending  on  the  last  page  with  a  printed 
or  illustrated  impression. 


Les  exemplaires  originaux  dont  la  couverture  wn 
papier  est  imprimde  sont  film^s  en  commenqant 
par  le  premier  plat  et  en  terminant  soit  par  la 
derniire  page  qui  comporte  une  empreinte 
d'impression  ou  d'illustration,  soit  par  le  second 
plat,  selon  le  cas.  Tous  les  autres  exemplaires 
originaux  sont  film^H  en  commenpant  par  la 
premidre  page  qui  cc.nporte  une  empreinte 
d'impression  ou  d'illustration  et  en  terminant  par 
la  dernidre  page  qui  comporte  une  telle 
empreinte. 


The  last  recorded  frame  on  each  microfiche 
shall  contain  the  symbol  •'^-(meaning  "CON- 
TINUED"), or  the  syribol  V  (meaning  "END"), 
whichever  applies. 


Un  des  symboles  suivants  apparaftra  sur  la 
dernidre  image  de  cheque  microfiche,  selon  ie 
cas:  le  symbols  — ►  signifie  "A  SUiVRE",  le 
symbole  V  signifie  "FIN". 


Maps,  plates,  charts,  etc.,  may  be  filmed  at 
different  reduction  ratios.  Those  too  large  to  be 
entirely  included  in  one  exposure  are  filmed 
beginning  in  the  upper  left  hand  corner,  left  to 
right  and  top  to  bottom,  as  many  frames  as 
required.  The  following  diagrams  illustrate  the 
method: 


Les  cartes,  planches,  tableaux,  etc.,  peuvnnt  Atre 
film6s  it  des  taux  de  reduction  diffirents. 
Lorsque  ie  document  est  trop  grand  pour  Atre 
reproduit  en  un  seul  clichA,  11  est  filmA  A  partir 
do  Tangle  supArieur  gauche,  de  gauche  A  droite, 
et  de  haut  en  bas,  en  prenant  le  nombre 
d'images  nAcessaire.  Les  diagrammes  suivants 
illustrent  la  mAthode. 


1 

2 

3 

:.    ,1, 

,       2 

3 

4 

5 

6 

/3. 


ON  THE  ORIGIN  OP  THE  SO-CALLED  "TEST- 
CELLS"  IN  THE  ASCIDIAN  OVUM.  By  J.  PLAY- 
FAIR  MoMURRICH,  B.  A.,  Assistant  in  the  Biological  Lahora- 
lory,  University  of  Toronto.     With  Plate  X. 


The  following  observations  have  been  made  in  the  hope  of 
elucidating  to  some  extent  the  nature  of  the  so-called  "  test-cells," 
so  characteristic  of  the  ova  of  Tunicates.  These  bodies  have  been 
described  by  various  authors  as  occurring  in  the  eggs  of  most  of 
the  commoner  forms,  and  under  normal  circumstances  probably 
do  not  make  their  appearance  until  after  fertilization.  Lacaze- 
Duthiers^^^  states  that  in  Molgula  a  true  layer  of  "test-cells"  is 
•wanting,  and  only  the  follicle-epithelium  surrounds  the  newly 
deposited  ovum.  Under  abnormal,  circumstances,  however,  they 
are  formed  at  a  much  earlier  period,  and  thus  in  most  eggs  that 
have  been  observed,  "  test-cells  "  were  present  or  soon  made  their 
appearance. 

My  observations  have  been  carried  on  for  the  most  part  on  ova 
of  Ascidia  amphora,  but  I  have  also  confirmed  most  of  them  by 
similar  experiments  on  eggs  of  Cynthia  ocellata.  I  made  use  only 
of  mature  or  almost  mature  eggs,  so  that  I  am  unable  to  give  as 
complete  an  account  of  some  points  as  could  be  desired. 

The  mature  eggs  oi  A.  amphora  (PI.  X,  Fig.  1)  have  an  average 
diameter  of  about  .255  mm.  and  present  on  optical  section  two  dis- 
tinctly marked  zones,  enclosing  a  semi-transparent  granular  mass, 
the  yolk.  The  outer  of  the  two  zones  is  formed  by  the  follicle- 
epithelium,  consisting  of  a  single  layer  of  cells  surrounding  the 
whole  egg,  and  presenting  on  a  surface  view  a  polygonal  appear- 
ance. On  examining  a  single  cell  which  has  become  separated 
from  the  egg,  with  a  rather  iiigh  magnifying  power,  its  interior  is 
seen  to  be  occupied  almost  entirely  by  a  number  of  vacuoles,  sepa- 
rated from  one  another  and  surrounded  by  very  delicate  bands  of 
protopla&m,  which,  in  some  of  the  angles  formed  by  the  meeting 
of  the  polygonal  vacuoles,  appear  as  dark  spots.  (PI.  X,  Fig.  2.) 
I  have  not  been  able  to  observe  the  development  of  these  cells,  but 
Semper ^^^  las  described  it  as  it  occurs  in  Molgula  nana,  where,  in. 

147 


148 


J.  PLAYFAIR  McMURRIGB. 


the  earliest  observed  stages,  they  appear  as  a  layer  of  flat  cells  on 
the  surface  of  the  egg,  which,  later  on,  become  prismatic,  and  in 
the  interior  of  which  2-4  yellow  granules  make  their  appear- 
ance. These  afterward  disappear  and  large  vacuoles  take  their 
place,  pressing  the  protoplasm  and  nucleus  to  one  side.  In  his 
figures,  the  formation  of  the  vacuoles  has  not  advanced  as  far  as  in 
the  eggs  I  studied,  but,  on  comparing  his  Figure  5,  Plate  I,  with 
my  Figure  2,  Plate  X,  it  will  at  once  be  recognized  that  the  ap- 
pearance I  have  observed  is  to  be  accounted  for  in  the  same 
manner,  the  vacuoles  having  become  exceedingly  abundant,  and 
pressed  the  original  contents  of  the  cell  to  the  periphery,  small 
portions  only  being  left  in  the  intervals  between  the  vacuoles. 

Fol,^^^  having  succeeded  in  tracing  tlie  origin  of  these  cells  still 
farther  back  in  Phallusia  intesiinalisy  states  that  they  are  not 
formed  from  the  ovary,  but  from  the  interior  of  the  egg  at  the 
boundary  between  the  yolk  and  the  nucleus,  and  wander  thence  to 
the  surface,  where  they  form  an  epithelial  layer  round  the  egg. 
One  would  fancy  at  first  that  the  eminent  observer  had  accidentally 
confused  "  test-cells  "  with  the  follicle-epithelium,  but  that  he  has 
not  done  so  is  evident  from  his  also  describing  the  "  test-cells  "  as 
formed  later  on.  This  discovery  is  of  great  interest,  both  from  its 
upsetting  all  former  theories  as  to  the  formation  of  these  cells, 
which  have  hitherto  always  been  considered  as  being  formed  from 
the  ovary,  and  also  from  the  singular  manner  in  which  Kowa- 
lewsky's  theory  in  regard  to  the  formation  of  the  "  test-cells  "  from 
these  cells  has  been  turned  upside  down,  these  bodies  {i.  e.  the 
**  test-cells")  being  formed  inlependently  from  the  yolk  (as  will  be 
seen  hereafter)  from  which  at  an  earlier  period  the  follicle-cells 
had  also  been  derived.  If  M.  Fol's  observations  are  correct,  it 
is  evident  that  the  term  "  follicle-cell "  is  entirely  a  misnomer, 
as  is  also  indeed  that  of  "  test-cell,"  both  being  to  a  high  degree 
misleading  to  one  who  has  not  studied  the  history  of  the  ap- 
pellations- 

Within  this  layer  of  so-called  "  follicle-cells"  comes  the  second 
izone  of  the  egg,  consisting  of  a  transparent,  apparently  homoge- 
neous structure,  which,  however,  when  acted  on  by  acetic  acid, 
becomes  markedly  granular.  This  is  the  egg-shell  or  "chorion" 
of  some  authors. 

In  the  interior  of  the  egg-shell  and  filling  it  almost  completely 
in  the  fresh  ovum,  is  the  yolk.    On  the  average  it  measured 


3r^6££t 


A8GIDIAN  OVA. 


149 


.236  mm,  and  was  of  a  yellowish  gray  color,  due  to  the  coloration 
of  the  constituent  granules.  In  the  majority  of  the  eggs  of  this 
Asoidian  I  examined,  no  nuclei  were  visible  either  in  the  fresh 
egg,  or  in  those  that  had  been  subjected  to  the  reaction  of  acetic 
acid  and  glycerine,  or  osmic  acid  and  carmine.  In  some,  however, 
a  clear  spot  was  noticeable,  usually  situated  eccentrically  (in  ore 
instance  at  the  periphery  of  the  egg),  and  measuring  .020-.08(5 
mm.  One  egg  presented  a  rather  peculiar  abnormality,  which  I 
deem  worthy  of  being  recorded.  The  peculiarity  consisted  in  the 
presence  of  two  distinct  nuclei,  both  situated  eccentrically  on  the 
same  side  of  the  egg  and  varying  somewhat  in  size,  the  larger 
measuring  .06  mm.  and  the  smaller  .04  mm.  I  am  certain  that  I 
did  not  observe  the  male  and  female  pronuclei,  as  the  egg  under 
observation  had  just  been  removed  from  the  ovary,  so  that  it  could 
not  have  been  impregnated  any  length  of  time,  if  at  all,  before 
my  observation  of  it. 

These  were  all  the  points  to  be  observed  in  a  perfectly  fresh 
ovum,  but  in  one  that  had  been  removed  from  the  ovary  for  a 
short  time,  or  which  had  been  subjected  to  the  action  of  various 
reagents,  there  was  to  be  seen  surrounding  the  yolk  a  layer  of 
bodies,  which  have  received  the  name  of  "  test-cells  "  from  the 
idea  that  they  were  the  cells  of  the  developing  ovum,  from  which, 
eventually,  the  characteristic  test  of  the  adult  Ascidian  was  formed. 
Kowalewsky  ^*^  in  his  paper  on  the  development  of  Aacidia  canina 
states  his  belief  that  such  is  the  fate  of  these  cells,  which,  he  also 
maintains,  have  their  origin  from  the  epithelial  cells  of  the  egg- 
follicle.  Later  on,  however,  in  his  paper  on  the  development  of 
Pyrosoma,^^^  he  withdraws  the  statement  that  the  mantle  is  formed 
from  the  "test-cells,"  but  still  adheres  to  the  opinion  that  these 
are  merely  follicle-epithelial  cells,  which  have  migrated  inwards 
towards  the  yolk.  Before  the  appearance  of  his  first  paper,  how- 
ever, KupflPer,^*^  after  investigating  the  subject,  came  to  the  conclu- 
sion that  the  "  test-cells  "  formed  at  the  surface  of  the  egg  itself, 
which  theory  had  been  independently  adopted  by  MetschnikoflP.^'^^ 
Before  the  publication  of  Kowalewsky's  second  papf-r,  Hertwig^*^ 
shewed  that  the  "  test-cells  "  take  no  part  in  the  formation  of  the 
mantle,  this  being  formed  as  a  secretion  of  a  homogeneous  substance 
from  the  epidermis,  into  which,  later  on,  cells  migrate  from  the 
epidermis.  Hertwig's  observations  were  made  on  PhaUuaia  mamil- 
IcUa  and  P.  virginea  (?),  and  have  been  confirmed  by  Semper ^^^  by 


150 


J.  PLATFAIB  McMUBBICK 


observations  on  Clavelina  vitrea  and  Cynthia  depresaa.  In  the 
same  year  that  Semper  published  his  observations,  a  paper  by 
TJssow^'^  appeared,  in  which  the  old  theories  first  advanced  by 
Kowalewsky  are  revived  and  most  emphatically  insisted  upon. 
He  says :  "  The  outer  mantle  of  the  Tunicates  is  developed,  not  as 
a  secretion  product  of  the  epidermal  cells  of  the  inner  mantle, 
(Hertwig,  Arsenjew,)  but  by  the  increase  in  number  and  growth  of 
the  so-called  'test-cells'  (Kupffer,  Kowalewski),"  and  again :  "The 
result  of  my  observations  on  the  formation  of  the  so-called  'test- 
cells'  is  in  complete  accord  with  that  of  A.  Kowalewski.  The 
yellow  bodies  are  in  fact  nothing  but  cells  of  the  Graafian 
follicle  . .  ;  .  " 

Semper  shews  that  in  the  several  species  in  which  he  examined 
the  ova,  the  "  test-cells  "  were  formed  in  the  interior  of  the  egg, 
and  that  by  the  action  of  reagents,  or  even  by  exposure  to  sea- 
water,  these  bodies  might  be  produced  in  immature  eggs.  He 
holds  that  they  are  devoid  of  a  nucleus  and  of  a  cell-wall,  and  dis- 
carding the  term  "test-cells,"  substitutes  instead  that  of  "test- 
drops."  ' 

My  own  observations  having  been  confined  to  mature  or  almost 
mature  eggs,  I  cannot  confirm  Professor  Semper's  statement  as  to 
the  production  of  these  peculiar  bodies  in  immature  eggs  by  means 
of  reagents,  but  these  have  the  effect  of  producing  them  in  most 
cases  almost  immediately  in  mature  eggs,  even  the  exposure  to  sea- 
water  for  a  short  time  being  sufficient  for  the  purpose.  Produced 
in  this  manner  these  bodies  are  :  naall  and  roundish  in  shape,  and 
in  their  interior  numerous  clear  highly-refractive  granules  are  to 
be  seen.  I  could  detect  no  nucleus  either  in  the  fresh  or  in  the 
stained  "drop,"  and  a  limiting  membrane  was  also  apparently 
wanting. 

As  regards  their  mode  of  origin  I  am  in  accord  with  the  obser- 
vations of  Kupffer,^*^  Metschinkoflr,^^^  etc.  When  an  egg  has  been 
removed  from  the  ovary  for  a  few  minutes,  there  appear  in  the 
interior  of  the  yolk,  numerous  clear  spots  situated  nearer  the 
periphery  than  the  centre.  In  no  case  did  they  make  their  ap- 
pearance at  the  centre  of  the  yolk,  and  though  in  Figure  3,  (PI.  X,) 
some  appear  to  be  very  close  to  it,  these  in  reality  are  peripheral 
and  appear  indistinctly  when  an  optical  section  of  the  egg  is  made 
and  accordingly  have  been  represented.  I  accordingly  conclude 
that  their  origin  is  peripheral  as  stated  by  MetschinkofiT.^^^     They 


\ 


'    v  * 


V* 


A8GIDIAN  OVA. 


tftl 


71 


f'radually  migrate  outwards,  until  they  form  a  layer  at  the  periph** 
ery  of  the  yolk  (PI.  X,  Fig.  4),  and  then  pass  outside  of  it  alto- 
gether. The  yolk  at  the  same  time  contracts  and  leaves  a  space 
between  its  circumference  and  the  egg-membrane,  in  which  the 
"test-cells"  lie,  forming  at  first  a  layer  round  the  yolk  (PI.  X, 
Fig.  6),  but  as  the  contraction  of  the  yolk  proceeds,  and  the  space 
becomes  larger,  they  move  away  from  the  surface  of  the  egg  and 
scatter  themselves  irregularly.  (PI.  X,  Fig.  6.) 

I  should  imagine  that  there  is  in  a  manner  a  separation  of  the 
egg  into  two  portions ;  an  outer,  consisting  of  protoplasm  with 
comparatively  few  yolk  granules,  and  an  inner,  containing  most  of 
the  yolk  granules  and  a  small  amount  of  protoplasm.  The  outer 
zone  is  of  no  further  use  in  the  process  of  development,  and  gradu* 
ally  splits  up  into  these  "test-drops,"  their  formation  commencing 
at  the  inner  part  of  the  zone  and  proceeding  outwards,  until  we 
have  numerous  "test-drops"  and  nothing  left  of  the  egg  but  a 
dense  mass  of  food-granules,  closely  packed  together  in  the  re* 
maining  protoplasm,  from  which  the  embryo  is  formed.  Metschino 
koff  ^'^  describes  this  separation  of  the  egg  into  two  portions.  He 
says :  "  In  the  greenish  protoplasm  of  a  young  egg  of  Asddia 
intestinalia,  fine  yolk-granules  collect  round  the  nucleus;  the 
number  of  these  becomes  continually  greater,  whereby  only  the 
peripheral  portion  of  the  protoplasm  retains  its  greenish  colora* 
tion.  This  layer  now  separates  itself  distinctly  from  the  central 
granular  portion  and  splits  up  into  a  great  number  of  round 
bodies  which  are  the  first  *  Tunic-cells.' "  From  this  description 
one  would  imagine  that  the  author  implied  that  the  "Tunic-cells'' 
were  formed  at  the  extreme  periphery  of  the  egg,  which,  however, 
is  not  the  case,  for  they  make  their  appearance  in  its  interior. 

On  treating  a  fresh  ovum  with  a  dilute  solution  of  acetic  acid 
(1  or  2  drops  of  commercial  acid  to  a  watch-glassful  of  water)  for 
about  half  an  hour,  its  appearance  becomes  considerably  changed. 
(PI.  X,  Fig.  6.)  The  interglobular  protoplasm  of  the  "  follicle- 
cells"  becomes  much  more  distinct,  and,  in  consequence,  the 
globules  themselves  become  more  plainly  marked  o^.  The  trans* 
parent,  apparently  homogeneous  egg-membrane  becomes,  as  men-' 
tioned  above,  distinctly  granular.  The  yolk  contracts  very  much, 
measuring  on  the  average  about  .116  mm.,  half  its  original  size. 
This  contraction  leaves  a  clear  space  between  the  yolk  and  the 
egg-membrane,  which,  however,  is  larger  in  one-half  of  its  oir» 
3 


152 


J.  PLAYFAIR  McMURRIGE. 


cumference  than  in  the  other,  owing  to  the  eccentric  position 
assumed  by  the  contracted  yolk.  In  this  clear  space  are  numerous 
"  test-cells,"  not  forming  a  layer  round  the  yolk,  as  they  usually 
do  in  an  egg  that  has  been  subjected  for  a  short  time  only  to  the 
action  of  acetic  acid  or  sea-water,  but  scattered  irregularly  around 
the  yolk.  The  "test-cells"  measure  .008  mm.  and  present  the 
appearance  described  above.  In  eggs  that  have  been  left  in  acetic 
acid  for  a  much  longer  period  (6-20  hours)  no  further  changes 
occur,  showing  that  the  acid  has  exerted  its  full  influence  on 
them. 

After  exposure  to  sea-water  for  six  hours,  very  much  the  same 
appearance  is  presented  as  with  dilute  acetic  acid.  The  "  follicle- 
cells,"  however,  shew  a  tendency  to  separate  from  the  egg-mem- 
brane, which,  on  its  part,  does  not  present  a  granular  appearance. 
(Pi.  X,  Fig.  7.)  . 

Upon  running  some  strong  picro-carmine  under  a  cover-glass, 
below  which  were  some  ova  in  sea-water,  very  important  changes 
occurred.  At  first  no  "test-cells"  were  to  be  seen,  but,  as  the 
picro-carmine  gradually  reached  the  egg,  and  the  picric  acid 
exerted  its  action  upon  it,  it  gradually  assumed  a  yellow  hue, 
whil'j,  at  the  same  time,  there  appeared  at  its  periphery  many 
small  spherical  bodies  of  a  round  or  oval  shape,  the  same  size  as 
the  "  test-cells,"  and  containing  in  their  interior  several  highly 
refractive  granules,  which,  in  fact,  render  them  apparent.  No 
"test-cells"  appear  outside  the  yolk,  which  retains  its  original  size. 
The  egg-membrane  assumes  a  pink  hue,  and,  after  some  time, 
becomes  distinctly  granular.  The  "follicle-cells"  do  not  stain  for 
some  time  and  show  a  tendency  to  separate  from  the  egg-mem- 
brane. (PI.  X,  Fig.  8.)  The  reaction  produced  by  very  dilute 
picro-carmine  is  also  rather  important.  After  being  subjected  to 
this  reagent  for  about  half  an  hour,  the  eggs  presented  ai.  appear- 
ance intermediate  between  that  produced  by  the  continued  action 
of  dilute  acetic  acid  and  that  following  the  employment  of  strong 
picro-carmine.  (PI.  X,  Fig.  9.)  The  yolk  contracts  to  a  slight 
degree,  and  "test-cells"  make  their  appearance,  filling  up  the 
small  space  between  the  paitly  contracted  yolk  and  the  egg- 
membrane. 

I  also  employed  osmic  acid  in  the  following  manner.  The  eggs 
were  placed  in  a  watch-glass  containing  sea-water,  to  which  1  or 
2  drops  of  I  per  cent,  osmio  acid  had  previously  been  added,  and 


<       > 


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I  I  i 


,  .v-^ 


<  p» 


ASOIDIAN  OVA. 


168 


allowed  to  remain  there  for  from  five  to  ten  minutes,  when  they 
were  removed  and  stained  with  Beale's  carmine.  In  most  cases 
no  change  occurred,  the  yolk  remaining  of  its  origii.il  size,  and  no 
"test-cells"  or  clear  spots  made  their  appearance  in  the  yolk,  with 
the  exception  of  one  instance,  in  which  I  did  perceive  a  number 
of  clear  spots  in  the  periphery  of  the  yolk. 

By  these  results  two  questions  are  suggested:  Ist.  What  are 
these  "  test-cells  ?  "  2d.  How  are  the  various  phenomena  caused 
by  the  various  reagents  to  be  explained  ?  I  shall  give  the  second 
question  priority.  The  explanation  that  seems  to  me  to  be  the 
simplest,  and  that  which  bears  the  stamp  of  probability  most  dis- 
tinctly, is,  that  these  phenomena  are  caused  by  the  varying  effects 
of  the  different  reagents  in  producing  a  contraction  of  the  proto- 
plasm of  the  yolk.  Thus,  osmic  acid,  which  "fixes"  the  proto- 
plasm immediately,  allows  of  little  or  no  contraction,  and  hence  no 
"test-cells"  appear;  with  picric  acid  (which  evidently  is  the  con- 
stituent of  the  picro-carmine  that  is  active  in  producing  the  phe- 
nomenon) a  slight  contraction  takes  place  before  the  protoplasm 
becomes  "fixed,"  whereby  the  "test-cells"  are  formed,  but  the 
contraction  is  not  sufficient  to  cause  them  to  pass  outside  the  yolk; 
and,  in  the  last  place,  with  acetic  acid  and  sea-water  there  is  no 
fixing  of  the  protoplasm,  and  the  contraction  goes  on  to  such  an 
extent  that  the  "test-cells"  are  driven  completely  outside  the 
yolk.  Strong  evidence  in  support  of  this  theory  is  afforded  by 
the  variation  in  the  action  of  picric  acid,  according  to  the  strength 
in  which  it  is  used.  For,  as  we  have  seen,  in  a  dilute  solution  so 
much  contraction  of  the  yolk  is  produced,  that  the  "test-cells"  do 
partly  pass  out.  ^        • 

Accordingly,  then,  the  "test-cells"  are  formed  by  a  contraction 
of  the  protoplasm  of  the  egg,  and  thus  we  can  readily  understand 
their  formation  in  a  developing  egg,  where  the  contraction  pro- 
duced by  the  process  of  cleavage  would  be  quite  sufficient  to  cause 
their  extrusion  from  the  yolk. 

We  are  now  in  a  position  to  discuss  the  question  as  to  the  nature 
of  these  "  test-cells."  Semper  ^^^  regards  them  as  merely  polar  glob- 
ules, comparing  them,  in  respect  to  their  number,  with  those  of 
the  Mollusca.  This  theory  is,  however,  untenable,  for  by  the  re- 
searches of  Hertwig  on  the  formation  of  the  polar  globules  in  the 
eggs  of  Hsemopis,  Nephelis,^'"^  Asteracanthion,  Mytilus,^"^  and 
other  forms,  we  know  that  the  polar  globules  are  formed  by  a  true 


154 


J.  PLAYFAIR  McMURBICH. 


cell-division,  and  arc  themselves  true  cells,  containing  a  nucleus, 
whereas  no  such  process  has  been  observed  during  the  formation 
of  the  "test-cells,"  and  I  for  my  part  am  sure  that  it  does  not  ob- 
tain, and,  as  Semper  himself  insists,  the  "test-cells"  are  not  true 
cells,  but  merely  "drops."  Fol,^**^  too,  states  that  in  Phallusia 
intestinalia  polar  globules  (two  in  number)  are  formed  after  the 
disappearance  of  the  original  nucleus  and  after  the  formation  of 
"test-cells."  Accordingly  then,  there  is  no  morphological  ho- 
mology between  the  polar  globules  and  the  "test-cells."  In  the 
eggs  of  certain  forms,  however,  such  as,  in  the  Amphibia,  Rana,^'"^ 
and  in  the  Pisces,  the  Trout,^'^>  after  the  disappearance  of  the  ger- 
minal vesicle,  peculiar  bodies  are  extruded  from  the  yolk  without 
any  spindle-formation  or  cell-division,  for  which  Hertwig  proposes 
the  name  of  excreted  bodies  (Excretkorper)  in  contradistinction  to 
the  polar  globules  formed  by  cell-division.  These  structures  have 
been  supposed  by  the  various  authors  to  be  the  remains  of  the  ger- 
minal vesicle,  and  thus,  as  far  as  their  mode  of  formation  is  con- 
cerned, probably  do  not  allo\7  of  comparison  with  the  "test-cells," 
but  since  they  resemble  these  latter  in  being  bodies  whose  presence 
in  the  egg  is  not  necessary  to  its  further  development,  and  since 
the  cause  of  their  appearance  is  evidently  the  same,  viz :  the  con- 
traction of  the  yolk  induced  by  a  stimulus,  I  think  there  can  be 
DO  objection  to  classifying  the  "test-cells"  with  them  as  Excret- 
korper. 

Wyville  Thomson,^'^^  however,  has  described  bodies  as  occurring 
in  Antedon  roaaoeua  which  bear  a  closer  homology  to  "test-cells" 
than  even  these  structures.  He  says :  "  Consequently  on  the  con- 
traction of  the  yolk,  a  number  of  minute  spherical  pale  yellow 
oil-globule£i  are  apparently  pressed  out  into  the  space  within  the 
Vitelline  membrane."  These  bodies  differ  from  "test-cells" 
only  in  the  fact  that  they  are  oil-globules,  whereas  "test-cells"  are 
distinctly  protoplasmic  in  their  nature,  and  contain  in  their  interior 
several  oil-globules  usually.  This  distinction,  however,  is  of  com- 
paratively little  moment,  and  both  in  their  mode  of  formation  and 
general  appearance  these  Excretkorper — for  so  they  also  may  be 
denominated — are  evidently  closely  related  to  "test-cells"  and 
.perhaps  identical  with  them. 

I  consider  these  "test-cells"  to  be  simply  masses  of  albuminous 
material  containing  two  or  three  granules  of  the  food-yolk,  and 
presume  that  they  are  in  reality  only  poriions  of  the  protoplasm 


A8CIDIAN  OVA. 


166 


of  the  egg,  which  have  been  forced  out  by  the  contraction.  If 
an  egg,  in  which  the  "test-cells"  have  passed  outside  the  yolk, 
be  subjected  to  pressure  sufficient  to  rupture  the  yolk-raembrane, 
allowing  the  yolk  to  come  into  contact  with  the  "test-cells,"  and 
at  the  same  time  leaving  the  egg-shell  intact,  the  "test-celis" 
commingle  completely  with  the  yolk  and  cannot  be  distingi^shed 
again.  The  granules  to  be  observed  in  a  "test-coll"  have  a  perfect 
resemblance,  both  in  shape  and  appearance,  to  those  remaining  in 
the  yolk  as  food,  so  that  it  may  be  presumed  that  they  are  in 
reality  the  same,  and  were  originally  situated  in  the  yolk,  in  that 
portion  of  the  protoplasm  which  formed  the  "  test-cell,"  and  were 
extruded  with  it.  • 

The  reason  why  portions  of  the  yolk,  originally  of  use  to  the 
embryo,  have  become  useless  and  are  extruded,  must  remain  unde- 
cided until  the  life-histories  of  more  of  the  lower  types  of  Ascidians 
have  been  fully  worked  out,  but  in  all  probability  the  explanation 
is  to  be  sought  for  in  a  change  in  the  life  of  an  ancestral  form, 
whereby  the  development  became  more  rapid  aufl  less  food-yolk 
was  required,  while,  at  the  same  time,  little  or  no  diminution  in 
the  amount  of  yolk  in  the  egg  was  produced. 


TABLE  OF  REFERENCES. 


1.  Lacaze-Duthiers.  Les  ascidies  simpler  des  c6le»  de  la  France. 
Arch,  de  zool.  exper.     Vol.  III.     1874.      . 

2.  Semper.  Ueher  die  Entstehung  der  geschichteten  cellulose- 
epidermis  der  Ascidien.  Arb.  aus  dem  zool.-zoot.  Inst,  zu  Wiirz- 
burg.     Bd.  II.     1875. 

8.  Fol.  Sur  la  formation  des  ceufs  chez  les  Aacidies.  Journ, 
de  Micographie.     T.  I.     1877. 

4.  Kowalev/sky.  Weitere  Studien  uber  die  Entwickelnng  der 
einfachen  Ascidien.    Arctiiv  fur  mikr.  Anat.     Bd.  VII.     1870. 

5.  Kowalewsk).  Ueber  die  Entwickelungsgeschichte  von  Pyro^ 
soma.    Archiv  fur  mikr.  Anat.    Bd.  XL     1875. 

6.  Kupflfer.  Die  Stammverwandtschaft  zwischen  Ascidien  und 
Wirbelthieren  nach  Untersuchungen  Uber  die  Entwichelung  der 
Ascidia  canina.     Archiv  fur  mikr.  Anat.    Bd.  VI.     1870. 


166 


J.  PLA7FAIR  McMUBRICH. 


T.  Metschinkoff.  Zur  Entioickelungsgeschichte  der  einfachen 
Ascidien.    Zeit.  fur  wissen.  Zool.     Bd.  XXII.     1872. 

8.  Hertwig.  Untersuchungen  iiber  den  Bau  und  die  Entwicke- 
lung  des  CelMlosemantels  der  Tunicaten.     Jen.  Zeit.    VII.     1872. 

9.  Ussow.  Zoologischembryologische  Untersuchungen:  Die 
Mantelthiere.    Archiv/ur  Naturg.    Jahrg.  XLI.     1875. 

10.  Hertwig.  Beitrdge  zur  Kennfnias  der  Bildung,  Befruchtung 
und  Theilung  des  thierischen  Eies.  2ter  Theil.  Morph.  Jahrb. 
Bd.  III.     1871 

1 1.  Hertwig.  Beitrage  zur  Kenntniss  der  Bildung,  Befruchtung 
und  Theilung  des  thierischen  Eies.  Ster  Theil.  Morph.  Jahrb. 
Bd.  IV.     1878. 

12.  Oellachcr.  Beitrage  zur  Entwickelungsgeschichte  der  Kno- 
chenfische.    Zeit.  fur  wissen.  Zool.     Bd.  XXII.     1872. 

13.  Wyville  Thomson.  On  the  Embryogeny  of  Antedon  rosaoeus, 
Jjinck  {Oomatula  rosacea  of  Lamarck).     Phil.  Trans.     1865. 


i 


i  ». 


EXPLANATION  OF  FIGURES. 


Figure  2 
'    drawn  with 

Figure  L— 

Figure  2.— 

Figure  3.— 

Figure  4.— 

Figure  5.— 

Figure  6.— 

«  Figure  7.— 
Figure  8.— 
Figure  9.- 


is  drawn  with  Hartnack  obj.  9,  oc.  2;  all  the  rest  are 
Hartnack  obj.  7,  oc.  2. 

-Fresh  egg  of  Ascidia  amphora. 

-Pomcle-cell.  ^  ' 

-Egg  after  short  exposure  to  sea<water.  "^ 

-Egg  after  longer  exposure  to  sea-water. 

-Egg  after  still  longer  exposure  to  sea-water. 

-Egg  after  exposure  for  half  an  hour  to  the  action  of  dilute 
acetic  acid. 

-Egg  after  exposure  to  sea-water  for  six  hours. 

-Egg  after  the  action  of  strong  picro- carmine. 

-Egg  after  the  action  of  very  dilute  picro-carmine. 


,»,< 


'   li 


STUDIES  FROM  BIOI,  LAB. 


VOT,   IT,  -  PLATE  X 


9 


rhr  HeUoty^^  Pnn*mo  Co  ^jyernortSi  ff/tstdn. 


